Drug metabolism is a change in a chemical structure of a compound caused by an enzymatic action in a living body. Such an enzym, contributing to drug metabolism, is called a drug metabolizing enzyme. Drug metabolizing enzymes are considered originally to catalyze synthetic reactions or decomposition reactions of biomolecules such as steroids, fatty acids, or bile acids, but can metabolize drugs administered to or invading the body (i.e., foreign substances), whereby the foreign substances are eliminated from the body.
A drug metabolizing reaction is basically composed of a phase I reaction and a phase II reaction. In the phase I reaction, one or more polar functional groups are introduced to a drug by oxidation, reduction, and/or hydrolysis. In the phase II reaction, one or more biomolecules such as glucuronic acid, sulfuric acid, or glutathione are bound to the functional group(s) generated in the phase I reaction. The phase I and phase II reactions impart an excellent water-solubility to the drug, and as a result, the drug is easily excreted from the body.
Metabolizing enzymes contributing to approximately 80% of all phase I drug metabolizing reactions are called “cytochrome P450” (hereinafter referred to as “CYP”). CYPs have a molecular weight of approximately 50000 and contain a protoheme as a prosthetic group. When an average molecular weight of an amino acid is regarded as 100, CYP is composed of approximately 500 amino acids. In the classification and nomenclature of CYPs, CYPs are systematically denoted by placing an Arabic numeral indicating a “family” and an alphabetic character indicating a “subfamily” after “CYP”. A group of CYP molecules showing a homology (amino acid sequence) of more than 40% is regarded as a family. A group of CYP molecules showing a homology of more than 55% is regarded as a subfamily. When a family contains two or more subfamilies, the subfamilies are denoted in alphabetical order (for example, CYP2A, CYP2B, and CYP2C). Plural CYP molecules contained in a subfamily are denoted by placing an additional Arabic numeral suffix (for example, CYP1A1). At present, Families 1 to 4 are known as drug-metabolism-type CYPs in mammals (non-patent reference 1).
As preferred conditions of dogs used as laboratory large animals, there may be mentioned, for example, (1) homogeneity in form, physiological response, or the like, (2) no genetical lack, and (3) clear birth records (such as parentage or date of birth). The most preferable variety meeting the above conditions is a beagle (non-patent reference 2). Therefore, a beagle is an animal variety widely used in a large animal test for safety and toxicity or pharmacokinetics when compounds are screened at the search stage. In this connection, canine CYPs contributing to a drug metabolizing mechanism in a beagle are gradually becoming clear. Some canine CYP families are identified by various cloning techniques. Full or partial sequences encoding CYP1A1 and CYP1A2 (non-patent reference 3), CYP2B11 (non-patent reference 4), CYP2C21 and CYP2C41 (non-patent reference 3 and non-patent reference 5), CYP2D15 (non-patent reference 6), CYP2E1 (non-patent reference 7), and CYP3A12 and CYP3A26 (non-patent reference 8 and non-patent reference 9) have been cloned. With respect to almost all of the CYPs described above, the full sequences containing an open reading frame (hereinafter referred to as ORF) have been determined, but the sequence encoding CYP1A2 has been only partially determined. A human CYP1 family includes two subfamilies A and B, which are induced by polycyclic aromatic hydrocarbons (such as 3-methylcholanthrene) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). The drug metabolizing mechanism in the CYP1 family is best maintained among CYPs, and thus substrate specificity in humans is very similar to that of laboratory animals. Oxidation of carcinogenic polycyclic aromatic hydrocarbons or mycotoxins, or hydroxylation of nitrogen atom(s) in aromatic amines or heterocyclic amines are typical reactions, and the CYP1 family is closely associated with a metabolic activation of a carcinogen (non-patent reference 10).
In a beagle, it has been confirmed that CYP1A1 is slightly expressed in lungs, but not expressed in the liver. CYP1A2 is expressed only in the liver, and accounts for approximately 4% of all CYPs expressed in the liver (non-patent reference 3 and non-patent reference 11).
An individual difference in drug responsivity is associated with that in a drug metabolizing ability. Until now, a difference in a drug metabolizing ability was found from a difference in internal dynamics. Tolbutamide, debrisoquine, and sparteine are typically known. It has been clarified that such a difference in internal drug dynamics is due to a difference in an activity of a metabolizing enzyme caused by single nucleotide polymorphism(s) [SNP(s)] in a CYP gene which metabolizes the drug. More particularly, when one or more bases among four kinds of bases contained in DNA are substituted, substitution of amino acid(s) in an enzyme protein, introduction of a stop codon into a DNA sequence, or a flame shift will occur. The substitution of amino acid(s) may sometimes reduce an enzyme activity, and the introduction of a stop codon and a flame shift may sometimes generate an immature protein. Therefore, drugs in which a remarkable individual difference in internal dynamics caused by SNP(s) in a drug metabolizing enzyme is exhibited, are known (non-patent reference 12).
As to SNPs in a human CYP1A2 gene, it was reported that an amount of CYP1A2 expressed was lowered by −3858G>A SNP (mutation CYP1A2*1C) and −164C>A SNP (mutation CYP1A2*1F) located in the upstream region of the gene. However, it was reported, as to −164C>A SNP, that an amount of CYP1A2 expressed was increased by an environmental factor, i.e., smoking, in comparison with genetic factors. Functions of other SNPs in the human CYP1A2 gene are not identified (non-patent reference 13, non-patent reference 14, and non-patent reference 15).
SNPs are most widely examined in CYPs, but new findings can be obtained from enzymes other than CYPs, or drug transporters. Thiopurine S-methyltransferase (hereinafter referred to as TPMT) is an enzyme which catalyzes methylation of some thiopurine drugs. TPMT is mainly located in the liver, but also is located in erythrocytes, and thus erythrocytes are used to analyze phenotypes in humans. When a TPMT activity in erythrocytes is used as an index, activities in whites exhibited a triphasic profile. A group exhibiting a high activity accounted for 88.6%, a group exhibiting a middle activity accounted for 11.1%, and a group exhibiting a low activity accounted for 0.3%. Evans et al. analyzed a TMPT gene in a leukemia patient with acute pancytopenia by 6-mercaptopurine, and revealed that the gene had three point mutations (TPMT*2, TPMT*3A, and TPMT*3C) with amino acid substitutions which caused the disappearance of the enzyme activity (non-patent reference 16). Salavaggione et al. examined a polymorphism in canine TPMT. A phenotype analysis revealed the same individual differences as those in humans, but no significant SNPs in the canine TPMT were found by a gene diagnosis (non-patent reference 17).
Further, some SNPs in a human MDR1 (multi drug resistance) gene encoding a drug transporter, P-glycoprotein (hereinafter referred to as P-gp) were reported. Hoffineyer et al. reported that Caucasian were analyzed, to find that a gene mutation from C to T at position 3435 of exon 26 in the MDR1 gene encoding P-gp reduced an amount of MDR1 expressed in the digestive tract and that a concentration of digoxin was increased in plasma after oral administration (non-patent reference 18). Mealey et al. revealed that differences of sensitivity to a macrolide antibiotic, ivermectin in a Collies central nervous system were due to immature P-gp generated by a frame shift mutation in the mdr1 gene (non-patent reference 19).
As to canine SNPs associated with drug metabolizing enzymes, Paulson et al. reported that beagles could be divided into an extensive metabolizer (hereinafter referred to as EM) group and a poor metabolizer (hereinafter referred to as PM) group with respect to a metabolic rate of a cyclooxygenase II inhibitor, celecoxib. It is thought that a CYP2D subfamily may contribute to the polymorphism, but the details are unknown (non-patent reference 20).
Miyashita et al. revealed that there are two groups of beagles having different patterns of metabolites from a phosphodiesterase IV inhibitor, 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidin-2(1H)-one in plasma (non-patent reference 21). Azuma et al. reported that beagles exhibited individual differences in a concentration of metabolites from an α7-nicotine acetylcholine receptor agonist, GTS-21 in plasma, and suggested that differences in an amount of CYP1A expressed might contribute to the individual differences (non-patent reference 22).
Similarly, Mise et al. reported that differences in an amount of CYP1A expressed in beagles caused individual differences in a concentration of an anti-benzodiazepine antagonist, AC-3933 in plasma (non-patent reference 23).
However, SNPs in canine CYPs which can clarify individual differences in a phenotype analysis of a drug metabolizing ability in beagles are not identified in a gene diagnosis.    (non-patent reference 1) Ryuich Kato and Tetsuya Kamataki ed., “YAKUBUTUTAISHAGAKU -IRYOUYAKUGAKU/DOKUSEIGAKU NOKISOTOSHITE-”, 2nd ed., TOKYOKAGAKUDOUZIN, Oct. 2000, p. 9-19    (non-patent reference 2) Hiroshi Otokawa, “INUNOSEIBUTUGAKU”, 1St ed., ASAKURASYOTEN, Sep. 1969, p. 179-187    (non-patent reference 3) “Molecular pharmacology”, U.S.A., 1990, Vol. 38, p. 644-651    (non-patent reference 4) “Archives of biochemistry and biophysics”, U.S.A., 1990, Vol. 281, p. 106-115    (non-patent reference 5) “Drug metabolism and disposition”, U.S.A., 1998, Vol. 26, p. 278-283    (non-patent reference 6) “Archives of biochemistry and biophysics”, U.S.A., 1995, Vol. 319, p. 372-382    (non-patent reference 7) “Drug metabolism and disposition”, U.S.A., 2000, Vol. 28, p. 98 1-986    (non-patent reference 8) “Biochimica et biophysica acta”, U.S.A., 1991, Vol. 1088, p. 319-322    (non-patent reference 9) “The journal of pharmacology and experimental therapeutics”, U.S.A., 1997, Vol. 283, p. 1425-1432    (non-patent reference 10) Ryuich Kato and Tetsuya Kamataki ed., “YAKUBUTUTAISHAGAKU -IRYOUYAKUGAKU/DOKUSEIGAKU NOKISOTOSHITE-”, 2nd ed., TOKYOKAGAKUDOUZIN, Oct. 2000, p.19-20    (non-patent reference 11) “Xenobiotica”, United Kingdom, 1996, Vol. 26, p. 755-763    (non-patent reference 12) Warner Kalow, Urs Meyer, and Rachel Tyndale ed., Tomohisa Ishikawa trans-ed., “PHARMACOGENOMICS -21 SEIKTNOSOUYAKUTOKONOIRYOU-”, 1st ed., TECHNOMICS, Dec. 2002, p. 29-43    (non-patent reference 13) “The Journal of Biochemistry”, Japan, 1999, Vol. 125, p. 803-808    (non-patent reference 14) “British journal of clinical pharmacology”, United Kingdom, 1999, Vol. 47, p. 445-449    (non-patent reference 15) “Pharmacogenetics”, U.S.A., 1999, Vol. 9, p. 367-375    (non-patent reference 16) Ryuich Kato and Tetsuya Kamataki ed., “YAKUBUTUTAISHAGAKU -IRYOUYAKUGAKU/DOKUSEIGAKU NOKISOTOSHITE-”, 2nd ed., TOKYOKAGAKUDOUZIN, Oct. 2000, p. 141-155    (non-patent reference 17) “Pharmacogenetics”, U.S.A., 2002, Vol. 12, p. 713-724    (non-patent reference 18) “Proceedings of the National Academy of Sciences of the United States of America”, U.S.A., 2000, Vol. 97, p. 3473-3478    (non-patent reference 19) “Pharmacogenetics”, U.S.A., 2001, Vol. 11, p. 722-733    (non-patent reference 20) “Drug metabolism and disposition”, U.S.A., 1999, Vol. 27, p. 1133-1142    (non-patent reference 21) “The 17th Annual Meeting of the Japanese Society for the Study of Xenobiotics, poster session”, Japan, 2002, 2OPE-46    (non-patent reference 22) “Drug metabolism and pharmacokinetics”, Japan, 2002, p. 75-82    (non-patent reference 23) “Drug metabolism and disposition”, U.S.A., 2004, Vol. 32, p. 240-245